BPS2026 – Cholesterol facilitates radical mediated and enzymatic lipid peroxidation

Lipid peroxidation (LPO) is a widely used marker of oxidative stress and contributes to aging and diverse diseases. Cholesterol, a major component of eukaryotic membranes, regulates lipid packing and fluidity but has also been associated with increased oxidative susceptibility. The molecular basis of cholesterol’s effect on LPO, however, remains incompletely understood. Here, we examined cholesterol-dependent oxidation pathways in large unilamellar vesicles using fluorescence-based assays. Oxidation kinetics showed that cholesterol enhanced both lipoxygenase (LOX) and cholesterol oxidase (ChOx) mediated reactions in a concentration-dependent manner. Mechanistic analysis indicated two complementary processes: LOX-derived lipid radicals reacted with cholesterol to form cholesterol hydroperoxides (Chol-OOH), whereas ChOx directly converted cholesterol into enone products. Cholesterol also influenced susceptibility to non-enzymatic oxidation. At moderate concentrations, enzymatic peroxidation facilitated penetration of reactive oxygen species, increasing oxidative reactions within membranes. At higher concentrations, spontaneous oxidation was detected even without added oxidants, consistent with reduced membrane stability and auto-oxidation. These results indicate that cholesterol concentration determines whether it primarily enhances enzymatic peroxidation or promotes spontaneous oxidative reactions. Overall, the data show that cholesterol functions not only as a structural lipid but also as an active participant in oxidative membrane chemistry. By modulating both enzymatic and non-enzymatic peroxidation pathways, cholesterol contributes directly to oxidative reactivity in membranes, providing a molecular basis for its role in cholesterol-dependent oxidative stress.